There is considerable evidence of strong tumor-promoting activity of weakly acidic phenolic fraction of tobacco smoke condensate (TSCPhFr) in polynuclear aromatic hydrocarbons (PAHs)-initiated animals. The identity of the tumor promoting phenolic component(s) in TSCPhFr is not known nor the mechanism(s) underlying its tumor-promoting activity. We observed TSCPhFr at non-cytotoxic concentrations attenuates ()-anti-benzo[a]pyrene-7,8- diol-9,10-epoxide [BPDE]-induced p53/NF-kB responses and promotes anchorage- independent cell growth (AICG) in JB6 cells. DNA damage-induced NF-kB activation has important role in p53- mediated apoptosis. We hypothesize that the tumor promoting activity of TSCPhFr is due to the abrogation of p53 accumulation and its function toward apoptosis in response to DNA damaging PAHs. Our studies include (1) purification of the tumor promoting component(s) from TSCPhFr using reverse phase HPLC, (2) determine the correspondence of inhibition of BPDE-induced p53 response and NFkB activation with apoptosis inhibition and increased AICG activity using chemical and genomic inhibitors, (3) if the findings in aim 2 above are affirmative then determine the effect of TSCPHFr on BPDE-induced p53-mediated NFkB activation (RAF/MAPK pathway), the expression of apoptosis regulatory proteins e.g. cIAP1, cIAP2, survivin and bcl-2 family proteins (Bax and bcl-2), degradation of PARP and activation of caspases, (4) if the findings in aim 2 above are negative, then in BPDE treated cells determine the effect of TSCPhFr on p73 response which is known to be involved in p53- independent apoptosis, and examine the correspondence of the inhibition of p73 response with AICG activity and inhibition of apoptosis using genomic inhibitor and (5) if inhibition of BPDE-induced p53, but not apoptosis, has correspondence with AICG activity then determine whether TSCPhFr can override BPDE-induced cell cycle arrest, inhibit p21WAF1 (p21) transactivation and up-regulate G1 cyclins (D1/D3/E) and associated cdk activities (cdk2, cdk4). In our studies we will use JB6 cl41 cells, a model cell line extensively used for the study of tumor promotion. The overall objective of the proposal is to identify the tumor promoting component/s in TSCPhFr and to understand the underlying mechanism(s) by which the phenolic component(s) elicits tumor promoting effect in PAH-initiated cells. Data obtained from these studies will help in assessing the associated health risk presented by tobacco smoke constituents and will be useful for therapeutic strategies in the prevention of cancer. The findings from studies undertaken in the project will open avenues for further detailed investigations on the mechanism of PAH-initiated tumor promotion by the phenolic fraction of TSC. Identification of tumor promoting phenolic component and understanding of the mechanism(s) by which phenolic component of tobacco smoke exerts its tumor promoting effect will help not only assessing the health risk presented by tobacco smoke constituents but also developing a strategy to eliminate the presence of particular tumor promoting phenolic component/s from tobacco leaf through chemical means or genetic manipulation. [unreadable] [unreadable] [unreadable]